Aim To establish a rapid method to efficiently iso-late mononuclear cells from central nervous system ( CNS) tis-sues of mice that can be effectively utilized for identification of various immune cell populations in a single sample by flow cy-tometry. Methods For defining the feasibility and practicality of the method, wild-type C57BL/6 mice and two mouse models of CNS disease including EAE mice and APP/PS1 mice were used in this study. After the collection and homogenization of the brain and spinal cord tissues respectively, the mononuclear cells were isolated by spinning the 70% -30% Percoll gradients. Cell activities were detected by trypan blue staining, and the im-mune population that infiltrated CNS was identified by flow cy-tometry. Results The results of trypan blue staining showed that the survival rate of the isolated cells was above 90% in all groups. Flow cytometry analysis showed that the relative num- bers of lymphocytes infiltrating CNS of EAE and AD mice in-creased significantly compared with wild-type C57BL/6 mice. In addition, the relative numbers of Th1 and Th17 cell subsets me-diating the inflammatory response also increased significantly, while the decreased regulatory T cells frequency was observed in the two mouse models of CNS disease. Conclusions The cells isolated by the 70% ~30% Percoll gradients centrifugation can be effectively utilized for the identification of various immune cell populations in a single sample by flow cytometry. The meth-od described in this article is simple and rapid in operation and with high survival rate and activity of the cells, which can be ap-plied to the study of the mononuclear cells in CNS.

OBJECTIVETo preliminarily investigate the long-term structural and functional injuries of mitochondria in rat brain caused by sepsis.

METHODSWistar rats were randomly assigned into sepsis and control groups. A rat model of sepsis was prepared by an intraperitoneal injection of 10 mg/kg lipopolysaccharide (LPS) of gram-negative bacteria, and the survival assay was performed. Eight rats in the sepsis group were sacrificed at 12, 24, 48, or 72 hours after LPS injection, while rats in the control group were sacrificed after an intraperitoneal injection of an equal volume of normal saline. Mitochondria were extracted from rat brain tissue. Mitochondrial membrane potential (MMP) and mitochondrial swelling level were determined by flow cytometry, and the activities of electron transport chain complexes (I-V) were measured using enzyme assay kits. Hematoxylin-eosin (HE) staining and electron microscopy were used to observe morphological changes in brain tissue and mitochondria.

RESULTSThe sepsis group had a significantly lower survival rate than the control group (P<0.01). The MMP and activities of electron transport chain complexes (I-V) in the sepsis group, which were significantly lower than those in the control group (P<0.05), were reduced to the lowest levels at 48 hours and partially recovered at 72 hours. The mitochondrial swelling level in the sepsis group, which was significantly higher than that in the control group (P<0.05), increased to the peak level at 48 hours and partially recovered at 72 hours. Hematoxylin and Eosin staining revealed substantial damages in the structure of brain tissue, and electron microscopy showed mitochondrial swelling, and vacuolization in a few mitochondria.

CONCLUSIONSIn the rat model of LPS-induced sepsis, both structural and functional injuries are found in cerebral mitochondria, and achieve the peak levels probably at around 48 hours.

Animals ; Brain ; pathology ; physiopathology ; ultrastructure ; Lipopolysaccharides ; toxicity ; Male ; Membrane Potential, Mitochondrial ; Mitochondria ; physiology ; ultrastructure ; Rats ; Rats, Wistar ; Sepsis ; chemically induced ; mortality ; physiopathology

Objective:To explore the role and mechanism of extracellular histones involved in lipopolysaccharide (LPS)-induced alveolar macrophage injury.Methods:The mouse alveolar macrophage cell line (MH-S) was cultured in vitro and passaged, and the cells were cultured to 80% of cells for cell proliferation. The cells were stimulated with 1 mg/L LPS for 3 hours and 50 mg/L exogenous histones for 3, 6, 12, and 24 hours, respectively (LPS+histones 3, 6, 12, 24 h groups), and other groups included phosphate buffered saline (PBS) control group (PBS group), LPS alone stimulation group (LPS group), the exogenous histones alone stimulation group (histones group) and heparin pretreatment histones group (heparin+LPS+histones group). The cells in each group were challenged with different reagent, the expression of lactate dehydrogenase (LDH) and inflammatory factors in the supernatant were detected by enzyme linked immunosorbent assay (ELISA), and the change of intracellular K + concentration was detected by FluxOR TMⅡgreen potassium channel. The proteins such as potassium channel protein (TWIK2), inflammasome (NLRP3), and apoptosis associated speck like protein containing a CARD (ASC) were determined by Western Blot. Results:Compared with the PBS group, the levels of LDH and inflammatory factors such as interleukin (IL-1β, IL-18) and tumor necrosis factor-α (TNF-α) were significantly increased after LPS stimulation group. Compared with the LPS group, the levels of LDH and inflammatory factors were significantly increased after the treatment with exogenous histones, and reached a peak after 3 hours of the histones stimulation [LDH (U/L): 123.10±1.83 vs. 85.32±1.66, IL-1β (mg/L): 40.75±2.60 vs. 18.78±1.37, IL-18 (mg/L): 49.94±2.45 vs. 30.19±1.82, TNF-α (mg/L): 36.51±1.56 vs. 20.84±1.61, all P < 0.01]. Western Blot results showed that compared with the LPS group, NLRP3, ASC and TWIK2 protein expression were significantly up-regulated in the LPS+histones group (NLRP3/GAPDH: 0.80±0.02 vs. 0.57±0.02, ASC/GAPDH: 0.57±0.02 vs. 0.38±0.01, TWIK2/GAPDH: 0.65±0.01 vs. 0.41±0.01, all P < 0.01), and the expression of the above proteins were significantly down-regulated after heparin pretreatment (NLRP3/GAPDH: 0.28±0.02 vs. 0.80±0.02, ASC/GAPDH: 0.25±0.02 vs. 0.57±0.02, TWIK2/GAPDH: 0.35±0.01 vs. 0.65±0.01, all P < 0.01), indicating that histones could activate NLRP3 through TWIK2 to participate in inflammatory reaction. In addition, intracellular K + concentration in LPS+histones group decreased significantly compared with the LPS group (fluorescence intensity: 35.48±2.53 vs. 83.92±3.11, P < 0.01). Compared with LPS+histones group, K + concentration increased significantly after pretreatment with heparin (fluorescence intensity: 72.10±1.78 vs. 35.48±2.53, P < 0.01), indicating that extracellular histones could cause K + massive efflux through TWIK2, and thus mediate NLRP3 activation and participate in inflammatory injury of alveolar macrophages. Conclusion:Extracellular histones can cause inflammatory damage in alveolar macrophages, and its mechanism may be related to the activation of NLRP3 by extracellular histones activation of TWIK2 channel to promote K + efflux.

Objective:To explore the mediating effect of physical activity on association between sedentary leisure-time and obesity indexes among hypertensive individuals.Methods:After excluding of those with a prior history of heart disease, stroke and cancer, a total of 20 178 hypertensive participants in the China Kadooire Biobank (CKB) study from Wuzhong district of Suzhou city were included. Mediating effect analysis was used to analyze the mediating effect of physical activity (PA) on correlation between sedentary leisure-time and body fat percentage (BFP), waist circumference (WC) and body mass index (BMI).Results:After adjusted for age, gender, smoking status, alcohol consumption, education levels, intake frequencies of meat and intake frequencies of fresh fruit, sedentary leisure-time (SLT) was negatively correlated with PA ( β=-0.246, P<0.001), but positively associated with BFP ( β=0.061, P<0.001), WC ( β=0.087, P<0.001) and BMI ( β=0.071, P<0.001). After including the mediator variable PA, the direct effect of SLT on obesity index was still significant. PA was negatively correlated with BFP, WC and BMI ( β=-0.052, -0.083 and -0.028, respectively, P<0.001). Analysis of mediating effect indicated that the association of SLT with BFP, WC and BMI were partly mediated by PA, the proportion of mediating effect was 20.820%, 23.421% and 9.915%. Stratified by gender, PA had mediating effect on SLT and all obesity indexes in women, while only on SLT and BFP and WC in men. Conclusions:There is a significant mediating effect of PA on correlation between SLT and obesity indexes among hypertensive individuals. Hypertensive patients should increase the level of physical activity and reduce sedentary behavior to achieve a profounder healthy effect.

Objective To observe the influences of Qingshen (Kidney-Clearing) Granules,with effects of clearing heat,resolving dampness and dispelling stasis,on expressions of Janus-activated kinase 2 (JAK2) and signal transducers and activator of transcriptions 3 (STAT3) in Janus-activated kinasesignaltransducer and activator of transcription (JAK/STAT) signaling pathway mediated by Leptin in patients with chronic renal failure (CRF)-damp-heat pattern,and investigate the mechanism of Qingshen Granules in treatment of CRF.Methods The patients with CRF-damp-heat pattern (n =68) were randomly divided into treatment group and control group (each n =34).The study was completed actually in 60 patients,and 29 in treatment group and 31 in control group.All 2 groups were given basic treatment of Western medicine,and treatment group was additionally given Qingshen Granules (thrice daily and 1 bas every time) for 12 weeks.The clinical efficacy and changes of serum creatinine (SCr),estimated glomerular filtration rate (eGFR),serum Leptin,JAK2 and STAT3 were observed in 2 groups.Results The clinical efficacy was 86.21％ (25/29) and total effective rate of TCM patterns was 82.76％ (24/29) in treatment group,and 58.06％ (18/31) and 54.84％ (17/31) in control group (P ＜0.05).After treatment,the level of SCr decreased significantly (P ＜ 0.01) and level of GFR increased significantly (P ＜ 0.01) in treatment group compared with those before treatment,which were superior to those in control group at the same term (P ＜ 0.05).After treatment,the level of Leptin,and expressions of JAK2 and STAT3 decreased significantly (P ＜ 0.01) in treatment group compared with those before treatment,which were superior to those in control group at the same term (P ＜ 0.05 or P ＜0.01).Conclusion Qingshen Granules can reduce the level of Leptin and expressions of JAK2 and STAT3,inhibit the activation of JAK/STAT signaling pathway mediated by Leptin,relieve clinical symptoms and improve kidney function in patients with CRF-damp-heat pattern.

Objective:To determine the association between recreational drug usage， high risk sexual behavior， and HIV infection among men who have sex with men（MSM） in Jinhua. Methods:A cross-sectional survey was conducted to recruit MSM for anonymous questionnaire survey and serological examination. We used EpiData3.1 for data entry and SPSS 19.0 for statistical analysis. Results:A total of 368 MSM were surveyed， in which the proportion of recreational drug usage was 13.3% （ 49 / 368 ）， with the HIV infection of 10.3% （38/ 368 ） and syphilis infection of 8.9% （25/368）. Risk factors associated with recreational drug usage were determined to be part-time job or being unemployed （OR=5.26； 95%CI： 2.10-13.18； P<0.001）， average monthly income ≥CNY 5 000 （OR=6.45； 95%CI： 2.87-14.61； P<0.001）， education level of high school or above （OR=1.56； 95%CI： 1.37-3.57； P=0.037）， sexual orientation being homosexual（OR=3.52； 95%CI： 1.60-7.33； P=0.002）， number of sexual partners >1 （OR=4.37； 95%CI： 1.76-10.82； P=0.001）， engaged in group sex （OR=7.90； 95%CI： 2.11-29.55； P=0.002） and previously diagnosed sexually transmitted diseases （OR=4.76； 95%CI： 1.29-17.65；P=0.019）. Conclusion:Prevalence of recreational drug usage among MSM in Jinhua is relatively low. MSM with part-time or unemployed status， monthly income ≥CNY 5 000， higher education level， homosexual orientation， multiple sexual partners， sexually transmitted diseases and group sex behavior are the risk factors associated with recreational drug usage in Jinhua city. Targeted countermeasures should be considered for intervention.

Objective:To assess the efficacy and safety of cinepazide maleate injection in the treatment of patients with acute ischemic stroke.Methods:A multicenter, randomized, double-blind, placebo-controlled phase Ⅳ clinical trial, led by Peking Union Medical College Hospital, was conducted in 65 Hospitals in China. The efficacy of cinepazide maleate injection in patients with acute anterior circulation cerebral infarction with onset time of ≤48 hours, 7≤National Institute of Health stroke scale (NIHSS) score ≤25 was assessed from August 2016 to February 2019, using the proportion of modified Rankin scale (mRS) score≤1 and Barthel index (BI) score≤95 on day 14 as efficacy endpoint. The patients were divided into treatment group who were treated with cinepazide maleate injection and control group who were treated with placebo.Results:A total 937 patients were involved in the final efficacy analysis (466 in treatment group and 471 in control group). The proportion of subjects with mRS score≤1 on day 14 after treatment were higher in the treatment group than that in the control group (102/466(21.89%) vs76/471(16.14%)). Logistic regression analysis showed that patients treated with cinepazide maleate were significantly more likely to have a favorable outcome (mRS score≤1) than patients treated with placebo on day 14 ( OR=0.677, 95% CI 0.484-0.948 , P=0.023), and patients treated with cinepazide maleate were more likely to reach independence in activities of daily living (Barthel Index ≥95) than those treated with placebo on day 14 (125/466(26.82%) vs 91/471(19.32%); OR=0.632, 95% CI0.459-0.869, P=0.005). The rate of adverse events was similar between the treatment and control groups. Conclusion:The 14-day treatment with cinepazide maleate injection could reduce the degree of disability whereas did not increase the risk of adverse events.

Objective:To analyze the sexual relationship power and its influencing factors in married couples with single human immunodeficiency virus （HIV） positive spouse， and to provide theoretical support for the development of appropriate HIV prevention and control measures. Methods:Data of HIV single positive couples were collected from the database of AIDS epidemic. Social demographic characteristics， AIDS related knowledge， sexual behavior and sexual relationship power of HIV single positive couples were obtained by self-designed questionnaire. Current situation and influencing factors of sexual relationship power of the negative spouses HIV single positive couples' were analyzed. Results:Among 191 HIV negative spouses， 72.3% had not heard of AIDS， 3.1% had a history of HIV testing， and 38.2% had high sexual power. Single factor analysis found that men （χ² =12.227，P<0.001）， high income （χ² =4.227，P =0.040）， high education level （χ² =18.188，P<0.001）， frequent drinking （χ²= 6.104，P=0.013）， and high-level awareness of AIDS （χ²=10.827，P=0.013） had higher power of sexual relationship. Multivariate analysis showed that men and individuals with college education or above had higher sexual power， with OR （95% CI） 4.102 （1.757-9.578） and 7.403 （2.810-19.503）， respectively. Conclusion:The low awareness rate of AIDS knowledge and the disharmonious relationship between husband and wife will increase the risk of AIDS transmission in the spouses. Male， economic income， education level and AIDS awareness rate are the important influencing factors of sexual relationship power.